The transmission of disease by blood products and other biological materials remains a serious health problem. While significant advances in blood donor screening and blood testing have occurred, viruses such as hepatitis B (HBV), hepatitis C (HCV), and human immunodeficiency virus (HIV) may escape detection in blood products during testing due to low levels of virus or viral antibodies. In addition to the viral hazard, there are currently no adequate, licensed tests to screen for the presence of non-viral microbes, such as bacteria or protozoans, in blood intended for use in transfusions. The risk also exists that a hitherto unknown pathogen may become prevalent in the blood supply and present a threat of disease transmission, as in fact occurred before the recognition of the risk of HIV transmission via blood transfusions.
Chemical agents have been introduced into blood or blood plasma to inactivate pathogens prior to clinical use of the blood product. Typically, for blood products having little or no red blood cell content, such as platelets and plasma, photochemically activated compounds such as psoralens are used. For red blood cell containing blood products, compounds have been developed for pathogen inactivation, which do not require photoactivation. These compounds typically have electrophilic groups that react with pathogens, more specifically with pathogen nucleic acid. For example, U.S. Pat. No. 5,055,485 describes the inactivation of viruses in cell and protein containing compositions using aryl diol epoxides. Other compounds that generate electrophiles in situ may be used. LoGrippo et al. evaluated the use of nitrogen mustard, CH3—N(CH2CH2Cl)2, for viral inactivation. LoGrippo et al., Proceedings of the Sixth Congress of the International Society of Blood Transfusion, Bibliotheca Haematologica (Hollander, ed.), 1958, pp. 225-230. More significantly, U.S. Pat. Nos. 6,410,219 and 5,691,132, the disclosures of which are hereby incorporated by reference, describe the use of compounds that have a nucleic acid targeting component as well as an electrophilic component that reacts with the nucleic acid in order to inactivate the pathogen. U.S. Pat. No. 6,514,987, the disclosure of which is hereby incorporated by reference, describes similar compounds, wherein the nucleic acid targeting component of the compound is linked to the reactive electrophilic component via a hydrolysable linker. U.S. Pat. Nos. 6,136,586 and 6,617,157, the disclosures of which are hereby incorporated by reference, describe using ethyleneimine oligomers and related compounds for pathogen inactivation. These ethyleneimine derived compounds typically have an aziridine group, which provides the reactive electrophilic component, and a polyamine component, which provides nucleic acid targeting of the compound. The general class of nucleic acid targeted compounds having an electrophilic or similar group reactive with the nucleic acid are used to inactivate pathogens in blood, blood products, and a variety of samples of biological origin.
There is some concern that, while these compounds are designed to react specifically with nucleic acids, they may still react with other components of the blood, such as proteins or cellular membranes. These side reactions are unfavorable, and may cause adverse effects, such as modifications of the proteins and cell membranes that may be recognized by the immune system. When such treated blood products are used repeatedly, they may result in an immune response of the recipient to the treated blood product. U.S. Pat. No. 6,709,810, the disclosure of which is hereby incorporated by reference, describes methods of quenching such pathogen inactivating compounds in order to reduce the level of any such adverse side reactions. However, while such methods significantly reduce unwanted side reactions, a further reduction of unwanted immune responses is desirable. Recent clinical trials using such compounds for the treatment of red blood cells have indicated the possibility of such adverse events. In a V.I. Technologies, Inc. press release dated Nov. 17, 2003, it was recommended that their Phase III chronic trial of the INACTINE™ Pathogen Reduction System for red blood cells be halted due to a concern with antibody responses to INACTINE™ treated red blood cells. In a Cerus Corporation press release dated Sep. 4, 2003, it was indicated they voluntarily halted a Phase III trial for their pathogen inactivated red blood cell program after two study patients developed antibodies to red blood cells treated with S-303, the compound used in their pathogen inactivation system for red blood cells. Such antibodies are typically detected with the use of an Indirect Anti-globulin Test that can be performed without a detailed knowledge of the nature or homogeneity of the actual antibody. Such assays are well known to those skilled to the art and are very sensitive, allowing the detection of as low as 500 molecules per RBC. The most common method of detecting these antibodies is through the mixing of patient sera with the RBC preparation that is a candidate for infusion and detecting whether an agglutination reaction occurs. This is called a cross match of the RBC unit to the patient serum. More sensitivity is provided by the inclusion of an anti-human immunoglobulin cross reacting with the antibody. This enhances the reactivity between IgGs or other antibodies on the surface of RBC. Finally, even more detection sensitivity can be obtained by the inclusion of potentiators in the reaction medium which enhance the on-rate of antibodies with one another (AABB manual 13th edition). Such assays are more sensitive than, for example, flow cytometry assays and may be observed even when other methods indicate the absence of any potential antibody. Such phenomena occur in clinical trials and many times are associated with specific patient populations that may have a higher tendency to develop these antibodies.
Thus, there is a need for methods to further reduce unwanted electrophilic side reactions of pathogen inactivating compounds that react with pathogens via an electrophilic group, while preserving the ability of the pathogen inactivating compound to inactivate harmful pathogens. Specifically, there is a need for improved methods of quenching pathogen inactivating compounds in red blood cells. Such a new method is needed to significantly reduce the risk of an adverse immune response to the red blood cells due to the treatment with a pathogen inactivating compound.